Analytical Guidelines for co-fractionation Mass Spectrometry Obtained through Global Profiling of Gold Standard Saccharomyces cerevisiae Protein Complexes.

Co-fractionation MS (CF-MS) is a technique with potential to characterize endogenous and unmanipulated protein complexes on an unprecedented scale. However this potential has been offset by a lack of guidelines for best-practice CF-MS data collection and analysis. To obtain such guidelines, this study thoroughly evaluates novel and published Saccharomyces cerevisiae CF-MS data sets using very high proteome coverage libraries of yeast gold standard complexes. A new method for identifying gold standard complexes in CF-MS data, Reference Complex Profiling, and the Extending 'Guilt-by-Association' by Degree (EGAD) R package are used for these evaluations, which are verified with concurrent analyses of published human data. By evaluating data collection designs, which involve fractionation of cell lysates, it is found that near-maximum recall of complexes can be achieved with fewer samples than published studies. Distributing sample collection across orthogonal fractionation methods, rather than a single high resolution data set, leads to particularly efficient recall. By evaluating 17 different similarity scoring metrics, which are central to CF-MS data analysis, it is found that two metrics rarely used in past CF-MS studies - Spearman and Kendall correlations - and the recently introduced Co-apex metric frequently maximize recall, whereas a popular metric-Euclidean distance-delivers poor recall. The common practice of integrating external genomic data into CF-MS data analysis is also evaluated, revealing that this practice may improve the precision and recall of known complexes but is generally unsuitable for predicting novel complexes in model organisms. If studying nonmodel organisms using orthologous genomic data, it is found that particular subsets of fractionation profiles (e.g. the lowest abundance quartile) should be excluded to minimize false discovery. These assessments are summarized in a series of universally applicable guidelines for precise, sensitive and efficient CF-MS studies of known complexes, and effective predictions of novel complexes for orthogonal experimental validation.

Twin enzymes, divergent control: The cholesterogenic enzymes DHCR14 and LBR are differentially regulated transcriptionally and post-translationally.

Cholesterol synthesis is a tightly regulated process, both transcriptionally and post-translationally. Transcriptional control of cholesterol synthesis is relatively well-understood. However, of the ∼20 enzymes in cholesterol biosynthesis, post-translational regulation has only been examined for a small number. Three of the four sterol reductases in cholesterol production, 7-dehydrocholesterol reductase (DHCR7), 14-dehydrocholesterol reductase (DHCR14), and lamin-B receptor (LBR), share evolutionary ties with a high level of sequence homology and predicted structural homology. DHCR14 and LBR uniquely share the same Δ-14 reductase activity in cholesterol biosynthesis, yet little is known about their post-translational regulation. We have previously identified specific modes of post-translational control of DHCR7, but it is unknown whether these regulatory mechanisms are shared by DHCR14 and LBR. Using CHO-7 cells stably expressing epitope-tagged DHCR14 or LBR, we investigated the post-translational regulation of these enzymes. We found that DHCR14 and LBR undergo differential post-translational regulation, with DHCR14 being rapidly turned over, triggered by cholesterol and other sterol intermediates, whereas LBR remained stable. DHCR14 is degraded via the ubiquitin-proteasome system, and we identified several DHCR14 and DHCR7 putative interaction partners, including a number of E3 ligases that modulate DHCR14 levels. Interestingly, we found that gene expression across an array of human tissues showed a negative relationship between the C14-sterol reductases; one enzyme or the other tends to be predominantly expressed in each tissue. Overall, our findings indicate that whereas LBR tends to be the constitutively active C14-sterol reductase, DHCR14 levels are tunable, responding to the local cellular demands for cholesterol.

Non-canonical ubiquitination of the cholesterol-regulated degron of squalene monooxygenase.

Squalene monooxygenase (SM) is a rate-limiting enzyme in cholesterol synthesis. The region comprising the first 100 amino acids, termed SM N100, represents the shortest cholesterol-responsive degron and enables SM to sense excess cholesterol in the endoplasmic reticulum (ER) membrane. Cholesterol accelerates the ubiquitination of SM by membrane-associated ring-CH type finger 6 (MARCH6), a key E3 ubiquitin ligase involved in ER-associated degradation. However, the ubiquitination site required for cholesterol regulation of SM N100 is unknown. Here, we used SM N100 fused to GFP as a model degron to recapitulate cholesterol-mediated SM degradation and show that neither SM lysine residues nor the N terminus impart instability. Instead, we discovered four serines (Ser-59, Ser-61, Ser-83, and Ser-87) that are critical for cholesterol-accelerated degradation, with MS analysis confirming Ser-83 as a ubiquitination site. Notably, these two clusters of closely spaced serine residues are located in disordered domains flanking a 12-amino acid-long amphipathic helix (residues Gln-62-Leu-73) that together confer cholesterol responsiveness. In summary, our findings reveal the degron architecture of SM N100, introducing the role of non-canonical ubiquitination sites and deepening our molecular understanding of how SM is degraded in response to cholesterol.

Control of Cognate Sense mRNA Translation by cis-Natural Antisense RNAs.

Cis-Natural Antisense Transcripts (cis-NATs), which overlap protein coding genes and are transcribed from the opposite DNA strand, constitute an important group of noncoding RNAs. Whereas several examples of cis-NATs regulating the expression of their cognate sense gene are known, most cis-NATs function by altering the steady-state level or structure of mRNA via changes in transcription, mRNA stability, or splicing, and very few cases involve the regulation of sense mRNA translation. This study was designed to systematically search for cis-NATs influencing cognate sense mRNA translation in Arabidopsis (Arabidopsis thaliana). Establishment of a pipeline relying on sequencing of total polyA+ and polysomal RNA from Arabidopsis grown under various conditions (i.e. nutrient deprivation and phytohormone treatments) allowed the identification of 14 cis-NATs whose expression correlated either positively or negatively with cognate sense mRNA translation. With use of a combination of cis-NAT stable over-expression in transgenic plants and transient expression in protoplasts, the impact of cis-NAT expression on mRNA translation was confirmed for 4 out of 5 tested cis-NAT:sense mRNA pairs. These results expand the number of cis-NATs known to regulate cognate sense mRNA translation and provide a foundation for future studies of their mode of action. Moreover, this study highlights the role of this class of noncoding RNAs in translation regulation.

Knockout of the Hmt1p Arginine Methyltransferase in Saccharomyces cerevisiae Leads to the Dysregulation of Phosphate-associated Genes and Processes.

Hmt1p is the predominant arginine methyltransferase in Saccharomyces cerevisiae Its substrate proteins are involved in transcription, transcriptional regulation, nucleocytoplasmic transport and RNA splicing. Hmt1p-catalyzed methylation can also modulate protein-protein interactions. Hmt1p is conserved from unicellular eukaryotes through to mammals where its ortholog, PRMT1, is lethal upon knockout. In yeast, however, the effect of knockout on the transcriptome and proteome has not been described. Transcriptome analysis revealed downregulation of phosphate-responsive genes in hmt1Δ, including acid phosphatases PHO5, PHO11, and PHO12, phosphate transporters PHO84 and PHO89 and the vacuolar transporter chaperone VTC3 Analysis of the hmt1Δ proteome revealed decreased abundance of phosphate-associated proteins including phosphate transporter Pho84p, vacuolar alkaline phosphatase Pho8p, acid phosphatase Pho3p and subunits of the vacuolar transporter chaperone complex Vtc1p, Vtc3p and Vtc4p. Consistent with this, phosphate homeostasis was dysregulated in hmt1Δ cells, showing decreased extracellular phosphatase levels and decreased total Pi in phosphate-depleted medium. In vitro, we showed that transcription factor Pho4p can be methylated at Arg-241, which could explain phosphate dysregulation in hmt1Δ if interplay exists with phosphorylation at Ser-242 or Ser-243, or if Arg-241 methylation affects the capacity of Pho4p to homodimerize or interact with Pho2p. However, the Arg-241 methylation site was not validated in vivo and the localization of a Pho4p-GFP fusion in hmt1Δ was not different from wild type. To our knowledge, this is the first study to reveal an association between Hmt1p and phosphate homeostasis and one which suggests a regulatory link between S-adenosyl methionine and intracellular phosphate.

A new link between transcriptional initiation and pre-mRNA splicing: The RNA binding histone variant H2A.B.

The replacement of histone H2A with its variant forms is critical for regulating all aspects of genome organisation and function. The histone variant H2A.B appeared late in evolution and is most highly expressed in the testis followed by the brain in mammals. This raises the question of what new function(s) H2A.B might impart to chromatin in these important tissues. We have immunoprecipitated the mouse orthologue of H2A.B, H2A.B.3 (H2A.Lap1), from testis chromatin and found this variant to be associated with RNA processing factors and RNA Polymerase (Pol) II. Most interestingly, many of these interactions with H2A.B.3 (Sf3b155, Spt6, DDX39A and RNA Pol II) were inhibited by the presence of endogenous RNA. This histone variant can bind to RNA directly in vitro and in vivo, and associates with mRNA at intron-exon boundaries. This suggests that the ability of H2A.B to bind to RNA negatively regulates its capacity to bind to these factors (Sf3b155, Spt6, DDX39A and RNA Pol II). Unexpectedly, H2A.B.3 forms highly decompacted nuclear subdomains of active chromatin that co-localizes with splicing speckles in male germ cells. H2A.B.3 ChIP-Seq experiments revealed a unique chromatin organization at active genes being not only enriched at the transcription start site (TSS), but also at the beginning of the gene body (but being excluded from the +1 nucleosome) compared to the end of the gene. We also uncover a general histone variant replacement process whereby H2A.B.3 replaces H2A.Z at intron-exon boundaries in the testis and the brain, which positively correlates with expression and exon inclusion. Taken together, we propose that a special mechanism of splicing may occur in the testis and brain whereby H2A.B.3 recruits RNA processing factors from splicing speckles to active genes following its replacement of H2A.Z.

MS2-Deisotoper: A Tool for Deisotoping High-Resolution MS/MS Spectra in Normal and Heavy Isotope-Labelled Samples.

High-resolution MS/MS spectra of peptides can be deisotoped to identify monoisotopic masses of peptide fragments. The use of such masses should improve protein identification rates. However, deisotoping is not universally used and its benefits have not been fully explored. Here, MS2-Deisotoper, a tool for use prior to database search, is used to identify monoisotopic peaks in centroided MS/MS spectra. MS2-Deisotoper works by comparing the mass and relative intensity of each peptide fragment peak to every other peak of greater mass, and by applying a set of rules concerning mass and intensity differences. After comprehensive parameter optimization, it is shown that MS2-Deisotoper can improve the number of peptide spectrum matches (PSMs) identified by up to 8.2% and proteins by up to 2.8%. It is effective with SILAC and non-SILAC MS/MS data. The identification of unique peptide sequences is also improved, increasing the number of human proteoforms by 3.7%. Detailed investigation of results shows that deisotoping increases Mascot ion scores, improves FDR estimation for PSMs, and leads to greater protein sequence coverage. At a peptide level, it is found that the efficacy of deisotoping is affected by peptide mass and charge. MS2-Deisotoper can be used via a user interface or as a command-line tool.

MethylQuant: A Tool for Sensitive Validation of Enzyme-Mediated Protein Methylation Sites from Heavy-Methyl SILAC Data.

The study of post-translational methylation is hampered by the fact that large-scale LC-MS/MS experiments produce high methylpeptide false discovery rates (FDRs). The use of heavy-methyl stable isotope labeling by amino acids in cell culture (heavy-methyl SILAC) can drastically reduce these FDRs; however, this approach is limited by a lack of heavy-methyl SILAC compatible software. To fill this gap, we recently developed MethylQuant. Here, using an updated version of MethylQuant, we demonstrate its methylpeptide validation and quantification capabilities and provide guidelines for its best use. Using reference heavy-methyl SILAC data sets, we show that MethylQuant predicts with statistical significance the true or false positive status of methylpeptides in samples of varying complexity, degree of methylpeptide enrichment, and heavy to light mixing ratios. We introduce methylpeptide confidence indicators, MethylQuant Confidence and MethylQuant Score, and demonstrate their strong performance in complex samples characterized by a lack of methylpeptide enrichment. For these challenging data sets, MethylQuant identifies 882 of 1165 true positive methylpeptide spectrum matches (i.e., >75% sensitivity) at high specificity (<2% FDR) and achieves near-perfect specificity at 41% sensitivity. We also demonstrate that MethylQuant produces high accuracy relative quantification data that are tolerant of interference from coeluting peptide ions. Together MethylQuant's capabilities provide a path toward routine, accurate characterizations of the methylproteome using heavy-methyl SILAC.

Characterization of the Interaction between Arginine Methyltransferase Hmt1 and Its Substrate Npl3: Use of Multiple Cross-Linkers, Mass Spectrometric Approaches, and Software Platforms.

This study investigated the enzyme-substrate interaction between Saccharomyces cerevisiae arginine methyltransferase Hmt1p and nucleolar protein Npl3p, using chemical cross linking/mass spectrometry (XL/MS). We show that XL/MS can capture transient interprotein interactions that occur during the process of methylation, involving a disordered region in Npl3p with tandem SRGG repeats, and we confirm that Hmt1p and Npl3p exist as homomultimers. Additionally, the study investigated the interdependencies between variables of an XL/MS experiment that lead to the identification of identical or different cross-linked peptides. We report that there are substantial benefits, in terms of biologically relevant cross-links identified, that result from the use of two mass-spectrometry-cleavable cross-linkers [disuccinimido sulfoxide (DSSO) and disuccinimido dibutyric urea (DSBU)], two fragmentation approaches [collision-induced dissociation and electron-transfer dissociation (CID+ETD)] and stepped high-energy collision dissociation (HCD)], and two programs (MeroX and XlinkX). We also show that there are specific combinations of XL/MS methods that are more successful than others for the two proteins investigated here; these are explored in detail in the text. Data are available via ProteomeXchange with identifier PXD008348.

Large Scale Mass Spectrometry-based Identifications of Enzyme-mediated Protein Methylation Are Subject to High False Discovery Rates.

All large scale LC-MS/MS post-translational methylation site discovery experiments require methylpeptide spectrum matches (methyl-PSMs) to be identified at acceptably low false discovery rates (FDRs). To meet estimated methyl-PSM FDRs, methyl-PSM filtering criteria are often determined using the target-decoy approach. The efficacy of this methyl-PSM filtering approach has, however, yet to be thoroughly evaluated. Here, we conduct a systematic analysis of methyl-PSM FDRs across a range of sample preparation workflows (each differing in their exposure to the alcohols methanol and isopropyl alcohol) and mass spectrometric instrument platforms (each employing a different mode of MS/MS dissociation). Through (13)CD3-methionine labeling (heavy-methyl SILAC) of Saccharomyces cerevisiae cells and in-depth manual data inspection, accurate lists of true positive methyl-PSMs were determined, allowing methyl-PSM FDRs to be compared with target-decoy approach-derived methyl-PSM FDR estimates. These results show that global FDR estimates produce extremely unreliable methyl-PSM filtering criteria; we demonstrate that this is an unavoidable consequence of the high number of amino acid combinations capable of producing peptide sequences that are isobaric to methylated peptides of a different sequence. Separate methyl-PSM FDR estimates were also found to be unreliable due to prevalent sources of false positive methyl-PSMs that produce high peptide identity score distributions. Incorrect methylation site localizations, peptides containing cysteinyl-S-ß-propionamide, and methylated glutamic or aspartic acid residues can partially, but not wholly, account for these false positive methyl-PSMs. Together, these results indicate that the target-decoy approach is an unreliable means of estimating methyl-PSM FDRs and methyl-PSM filtering criteria. We suggest that orthogonal methylpeptide validation (e.g. heavy-methyl SILAC or its offshoots) should be considered a prerequisite for obtaining high confidence methyl-PSMs in large scale LC-MS/MS methylation site discovery experiments and make recommendations on how to reduce methyl-PSM FDRs in samples not amenable to heavy isotope labeling. Data are available via ProteomeXchange with the data identifier PXD002857.

Novel N-terminal and Lysine Methyltransferases That Target Translation Elongation Factor 1A in Yeast and Human.

Eukaryotic elongation factor 1A (eEF1A) is an essential, highly methylated protein that facilitates translational elongation by delivering aminoacyl-tRNAs to ribosomes. Here, we report a new eukaryotic protein N-terminal methyltransferase, Saccharomyces cerevisiae YLR285W, which methylates eEF1A at a previously undescribed high-stoichiometry N-terminal site and the adjacent lysine. Deletion of YLR285W resulted in the loss of N-terminal and lysine methylation in vivo, whereas overexpression of YLR285W resulted in an increase of methylation at these sites. This was confirmed by in vitro methylation of eEF1A by recombinant YLR285W. Accordingly, we name YLR285W as elongation factor methyltransferase 7 (Efm7). This enzyme is a new type of eukaryotic N-terminal methyltransferase as, unlike the three other known eukaryotic N-terminal methyltransferases, its substrate does not have an N-terminal [A/P/S]-P-K motif. We show that the N-terminal methylation of eEF1A is also present in human; this conservation over a large evolutionary distance suggests it to be of functional importance. This study also reports that the trimethylation of Lys(79) in eEF1A is conserved from yeast to human. The methyltransferase responsible for Lys(79) methylation of human eEF1A is shown to be N6AMT2, previously documented as a putative N(6)-adenine-specific DNA methyltransferase. It is the direct ortholog of the recently described yeast Efm5, and we show that Efm5 and N6AMT2 can methylate eEF1A from either species in vitro. We therefore rename N6AMT2 as eEF1A-KMT1. Including the present work, yeast eEF1A is now documented to be methylated by five different methyltransferases, making it one of the few eukaryotic proteins to be extensively methylated by independent enzymes. This implies more extensive regulation of eEF1A by this posttranslational modification than previously appreciated.

Protein substrates of the arginine methyltransferase Hmt1 identified by proteome arrays.

Arginine methylation on nonhistone proteins is associated with a number of cellular processes including RNA splicing, protein localization, and the formation of protein complexes. In this manuscript, Saccharomyces cerevisiae proteome arrays carrying 4228 proteins were used with an antimethylarginine antibody to first identify 88 putatively arginine-methylated proteins. By treating the arrays with recombinant arginine methyltransferase Hmt1, 42 proteins were found to be possible substrates of this enzyme. Analysis of the putative arginine-methylated proteins revealed that they were predominantly nuclear or nucleolar in localization, consistent with the localization of Hmt1. Many are involved in known methylarginine-associated functions, such as RNA processing and ribonucleoprotein complex biogenesis, yet others are of newer classes, namely RNA/DNA helicases and tRNA-associated proteins. Using ex vivo methylation and MS/MS, a set of 12 proteins (Brr1, Dia4, Hts1, Mpp10, Mrd1, Nug1, Prp43, Rpa43, Rrp43, Spp381, Utp4, and Npl3), including the RNA helicase Prp43 and tRNA ligases Dia4 and Hts1, were all validated as Hmt1 substrates. Interestingly, the majority of these also had human orthologs, or family members, that have been documented elsewhere to carry arginine methylation. These results confirm arginine methylation as a widespread modification and Hmt1 as the major arginine methyltransferase in the S. cerevisiae cell.

Yeast proteins Gar1p, Nop1p, Npl3p, Nsr1p, and Rps2p are natively methylated and are substrates of the arginine methyltransferase Hmt1p.

The Hmt1 methyltransferase is the predominant arginine methyltransferase in Saccharomyces cerevisiae. There are 18 substrate proteins described for this methyltransferase, however native sites of methylation have only been identified on two of these proteins. Here we used peptide immunoaffinity enrichment, followed by LC-ETD-MS/MS, to discover 21 native sites of arginine methylation on five putative Hmt1 substrate proteins, namely Gar1p (H/ACA ribonucleoprotein complex subunit 1), Nop1p (rRNA 2'-O-methyltransferase fibrillarin), Npl3p (nucleolar protein 3), Nsr1p (nuclear localization sequence-binding protein), and Rps2p (40S ribosomal protein S2). The sites, many of which were found to be mono- or di-methylated, were predominantly found in RGG (Arg-Gly-Gly) motifs. Heavy methyl-SILAC validated the majority of these peptides. The above proteins, and relevant sites of methylation, were subsequently validated by in vitro methylation with recombinant Hmt1. This brings the total of Hmt1 substrate proteins for which native methylation sites have been identified to five.

Lysine methylation modulates the protein-protein interactions of yeast cytochrome C Cyc1p.

In recent years, protein methylation has been established as a major intracellular PTM. It has also been proposed to modulate protein-protein interactions (PPIs) in the interactome. To investigate the effect of PTMs on PPIs, we recently developed the conditional two-hybrid (C2H) system. With this, we demonstrated that arginine methylation can modulate PPIs in the yeast interactome. Here, we used the C2H system to investigate the effect of lysine methylation. Specifically, we asked whether Ctm1p-mediated trimethylation of yeast cytochrome c Cyc1p, on lysine 78, modulates its interactions with Erv1p, Ccp1p, Cyc2p and Cyc3p. We show that the interactions between Cyc1p and Erv1p, and between Cyc1p and Cyc3p, are significantly increased upon trimethylation of lysine 78. This increase of interaction helps explain the reported facilitation of Cyc1p import into the mitochondrial intermembrane space upon methylation. This first application of the C2H system to the study of methyllysine-modulated interactions further confirms its robustness and flexibility.

The terminal enzymes of cholesterol synthesis, DHCR24 and DHCR7, interact physically and functionally.

Cholesterol is essential to human health, and its levels are tightly regulated by a balance of synthesis, uptake, and efflux. Cholesterol synthesis requires the actions of more than twenty enzymes to reach the final product, through two alternate pathways. Here we describe a physical and functional interaction between the two terminal enzymes. 24-Dehydrocholesterol reductase (DHCR24) and 7-dehydrocholesterol reductase (DHCR7) coimmunoprecipitate, and when the DHCR24 gene is knocked down by siRNA, DHCR7 activity is also ablated. Conversely, overexpression of DHCR24 enhances DHCR7 activity, but only when a functional form of DHCR24 is used. DHCR7 is important for both cholesterol and vitamin D synthesis, and we have identified a novel layer of regulation, whereby its activity is controlled by DHCR24. This suggests the existence of a cholesterol "metabolon", where enzymes from the same metabolic pathway interact with each other to provide a substrate channeling benefit. We predict that other enzymes in cholesterol synthesis may similarly interact, and this should be explored in future studies.

MicroRNA Regulatory Mechanisms Play Different Roles in Arabidopsis.

Plant microRNAs (miRNAs) operate by guiding the cleavage or translational inhibition of mRNA targets. They act as key gene regulators for development and environmental adaptation, and Dicer-partnering proteins DRB1 and DRB2 govern which form of regulation plays the dominant role. Mutation of Drb1 impairs transcript cleavage, whereas mutation of Drb2 ablates translational inhibition. Regulation of gene expression by miRNA-guided cleavage has been extensively studied, but there is much less information about genes regulated through miRNA-mediated translation inhibition. Here, we compared the proteomes of drb1 and drb2 mutants to gain insight into the indirect effect of the different miRNA regulatory mechanisms in Arabidopsis thaliana. Our results show that miRNAs operating through transcript cleavage regulate a broad spectrum of processes, including catabolism and anabolism, and this was particularly obvious in the fatty acid degradation pathway. Enzymes catalyzing each step of this pathway were upregulated in drb1. In contrast, DRB2-associated translational inhibition appears to be less ubiquitous and specifically aimed toward responses against abiotic or biotic stimuli.

Proteogenomic Discovery of a Small, Novel Protein in Yeast Reveals a Strategy for the Detection of Unannotated Short Open Reading Frames.

In recent years, proteomic data have contributed to genome annotation efforts, most notably in humans and mice, and spawned a field termed "proteogenomics". Yeast, in contrast with higher eukaryotes, has a small genome, which has lent itself to simpler ORF prediction. Despite this, continual advances in mass spectrometry suggest that proteomics should be able to improve genome annotation even in this well-characterized species. Here we applied a proteogenomics workflow to yeast to identify novel protein-coding genes. Specific databases were generated, from intergenic regions of the genome, which were then queried with MS/MS data. This suggested the existence of several putative novel ORFs of <100 codons, one of which we chose to validate. Synthetic peptides, RNA-Seq analysis, and evidence of evolutionary conservation allowed for the unequivocal definition of a new protein of 78 amino acids encoded on chromosome X, which we dub YJR107C-A. It encodes a new type of domain, which ab initio modeling suggests as predominantly α-helical. We show that this gene is nonessential for growth; however, deletion increases sensitivity to osmotic stress. Finally, from the above discovery process, we discuss a generalizable strategy for the identification of short ORFs and small proteins, many of which are likely to be undiscovered.

Proteomic Validation of Transcript Isoforms, Including Those Assembled from RNA-Seq Data.

Human proteome analysis now requires an understanding of protein isoforms. We recently published the PG Nexus pipeline, which facilitates high confidence validation of exons and splice junctions by integrating genomics and proteomics data. Here we comprehensively explore how RNA-seq transcriptomics data, and proteomic analysis of the same sample, can identify protein isoforms. RNA-seq data from human mesenchymal (hMSC) stem cells were analyzed with our new TranscriptCoder tool to generate a database of protein isoform sequences. MS/MS data from matching hMSC samples were then matched against the TranscriptCoder-derived database, along with Ensembl and the neXtProt database. Querying the TranscriptCoder-derived or Ensembl database could unambiguously identify ∼450 protein isoforms, with isoform-specific proteotypic peptides, including candidate hMSC-specific isoforms for the genes DPYSL2 and FXR1. Where isoform-specific peptides did not exist, groups of nonisoform-specific proteotypic peptides could specifically identify many isoforms. In both the above cases, isoforms will be detectable with targeted MS/MS assays. Unfortunately, our analysis also revealed that some isoforms will be difficult to identify unambiguously as they do not have peptides that are sufficiently distinguishing. We covisualize mRNA isoforms and peptides in a genome browser to illustrate the above situations. Mass spectrometry data is available via ProteomeXchange (PXD001449).

Polymer-albumin conjugate for the facilitated delivery of macromolecular platinum drugs.

The delivery of macromolecular platinum drugs into cancerous cells is enhanced by conjugating the polymer to albumin. The monomers N-(2-hydroxypropyl)methacrylamide (HPMA) and Boc protected 1,3-diaminopropan-2-yl acrylate (Ac-DAP-Boc) are copolymerized in the presence of a furan protected maleimide functionalized reversible addition-fragmentation chain transfer (RAFT) agent. The resulting polymer with a composition of P(HPMA14 -co-(Ac-DAP-Boc)9 ) and a molecular weight of Mn = 7600 g mol(-1) (D = 1.24) is used as a macromolecular ligand for the conjugation to the platinum drug. Thermogravimetric analysis reveals full conjugation. After deprotection of the maleimide functionality of the polymer, the reactive polymer is conjugated to albumin using the Cys34 functionality. The conjugation is monitored using size exclusion chromatography, MALDI-TOF (matrix assisted laser desorption ionization time-of-flight), and SDS Page (sodium dodecyl sulphate polyacrylamide gel electrophoresis). The polymer-albumin conjugates self-assemble in water into nanoparticles of sizes of around 80 nm thanks to the hydrophobic nature of the platinum drugs. The albumin coated nanoparticles are readily taken up by ovarian cancer cell lines and they show superior toxicity compared to a control sample without protein coating.

Interactions affected by arginine methylation in the yeast protein-protein interaction network.

Protein-protein interactions can be modulated by the methylation of arginine residues. As a means of testing this, we recently described a conditional two-hybrid system, based on the bacterial adenylate cyclase (BACTH) system. Here, we have used this conditional two-hybrid system to explore the effect of arginine methylation in modulating protein-protein interactions in a subset of the Saccharomyces cerevisiae arginine methylproteome network. Interactions between the yeast hub protein Npl3 and yeast proteins Air2, Ded1, Gbp2, Snp1, and Yra1 were first validated in the absence of methylation. The major yeast arginine methyltransferase Hmt1 was subsequently included in the conditional two-hybrid assay, initially to determine the degree of methylation that occurs. Proteins Snp1 and Yra1 were confirmed as Hmt1 substrates, with five and two novel arginine methylation sites mapped by ETD LC-MS/MS on these proteins, respectively. Proteins Ded1 and Gbp2, previously predicted but not confirmed as substrates of Hmt1, were also found to be methylated with five and seven sites mapped respectively. Air2 was found to be a novel substrate of Hmt1 with two sites mapped. Finally, we investigated the interactions of Npl3 with the five interaction partners in the presence of active Hmt1 and in the presence of Hmt1 with a G68R inactivation mutation. We found that the interaction between Npl3 and Air2, and Npl3 and Ded1, were significantly increased in the presence of active Hmt1; the interaction of Npl3 and Snp1 showed a similar degree of increase in interaction but this was not statistically significant. The interactions of Npl3 and Gbp2, along with Npl3 and Yra1, were not significantly increased or decreased by methylation. We conclude that methylarginine may be a widespread means by which the interactions of proteins are modulated.